We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale for this strategy is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), viruses that model HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. In studying vaccine-induced cellular immunity, we have recently focused on several specific cell types. Natural killer (NK) cells are a key population which have a rapid response potential, can kill target cells directly, and also mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC) which has been shown to contribute to protective efficacy. They also have recently been shown to persist for long periods of time in vivo and to have the capacity to establish immunologic memory. We have studied the phenotype and function of circulatory and tissue-resident NK cells in a unique cohort of SIV controlling rhesus macaques that maintained low to undetectable levels of viremia during the chronic phase of infection. We compared the NK responses of these macaques with those observed in SIV non-controlling and uninfected macaques, aiming to identify markers and activities of NK subpopulations associated with disease control. Most of the differences among the NK cells of the three groups of macaques were seen in tissue-resident cells. While SIV infection resulted in NK cell dysfunction, double negative NK cells (CD56-CD16-), and those expressing CXCR3, NKG2D, and IL-18Ralpha were associated with viremia control, as was antibody-dependent cytotoxic function. Double-negative NK cells have been reported to have potent cytotoxic activity and IFN-gamma production. The results of this study suggested several novel targets for therapeutic intervention. Currently we are studying the effect of vaccination on NK cells, particularly those in mucosal tissues, and investigating whether subsets of mucosal NK cells are associated with protective efficacy of our vaccine regimen. In HIV infection, CD8+ T cells play a critical role in controlling viremia. B cell follicles have been thought to be immune privileged sites of HIV infection. However, follicular CD8+ T cells (fCD8 cells) that reside within B cell follicles were recently described. We have observed comparable levels of fCD8 cells between chronically SIV infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. We investigated the role of SIV-specific fCD8 cells in lymph nodes (LNs) over the course of SIV infection in rhesus macaques. fCD8, T follicular helper (Tfh), and T follicular regulatory (Tfreg) cells were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than HVL animals, and their cell frequencies negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfregs via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to negatively correlate, and a positive correlation was seen between Tfreg cell number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Overall, our results suggested that elimination of virus-infected cells in B cell follicles does not only depend on the cytotoxicity of fCD8 cells but also on interaction of fCD8 cells with other T follicular cells including Tfh and Tfreg. Future studies will be critical in determining if immunization can induce potent fCD8 cells leading to protection or enhanced control of viremia. Further understanding of how to direct the differentiation and function of fCD8 and other follicular T cell populations should facilitate design of new strategies for eliminating HIV/SIV reservoirs. Vaccine strategies targeting the earliest stages of infection at the mucosal portal of entry may prevent HIV acquisition and systemic infection. Macrophages are one of the first cells that are encountered by HIV during vaginal exposure. Early recruitment of macrophages to the cervical mucosa can induce chemokine expression that promotes recruitment of additional susceptible macrophages and CD4+ T-cells, facilitating establishment of infection and viral dissemination. We evaluated early responses of mucosal macrophages to our replicating adenovirus recombinant vaccine. Rather than changes in chemokine expression that would facilitate infection, our results showed a vaccine-induced increase in CCL3 expression which can block access to and down-regulate the SIV co-receptor CCR5, and a reduction in recruitment of susceptible CD4+ T cells. Thus, use of this vaccine vector may contribute to decreased viral acquisition upon virus exposure. Dendritic cells (DCs) initiate and control responses of most effector immune cells. We investigated DC populations following vaccination with our replicating Adenovirus-recombinant vaccine in order to better understand cellular mechanisms elicited by mucosal antigen expression. Plasmacytoid DCs (pDCs) and Langerhans cells (LCs) were significantly increased in the rectal mucosa following immunization. However, in blood, pDCs tended to decrease while myeloid DCs (mDCs) significantly increased. Regardless of frequencies and location, all DC subsets showed up-regulated expression of activation markers and a lymph node migration marker. Pro-inflammatory cytokines and B cell-activating factor were also produced. No significant differences in DC activation were seen between the vaccine and control groups (the latter received empty Adenovirus vector) indicating the DCs were responding to the vector rather than the inserted viral genes. Antigen-specific memory T cells were increased in both rectal mucosa and blood after immunizations. Following immunization, rectal pDCs and mDCs were capable of inducing proliferation of autologous naive lymphocytes, especially CD8 T cells, and the vaccine group showed higher levels of T cell proliferation than controls after in vitro Env protein stimulation. Our results highlight the rapid response and potential roles of DCs in mucosal immune activation after replicating adenovirus immunization and identify the initial cellular mechanisms of the replicating adenovirus vaccine regimen in the rhesus macaque model.